RNA-based therapeutics offer a powerful paradigm for treating disease by targeting heretofore ?undruggable? genes, allowing highly specific silencing of pathologic gene expression to heal heretofore hopeless illnesses. However, a safe and efficient method for targeted delivery of cell-impermeant RNA drugs has remained elusive. A major hurdle for RNA-based therapies using vascular delivery is to circumvent the endothelial barrier. We have been developing a unique technology using intravenously injected RNA-loaded microbubbles (MB) which are triggered to cavitate by ultrasound (US), causing transient permeabilization of the adjacent cell membrane and endocytosis-independent uptake of the RNA by extravascular target cells. The potential of this site-specific, non- invasive delivery method is extra-ordinary, more so because the MBs and US transducer also confer capability for simultaneous real-time image-guided therapy. Despite its pre-clinical proof of concept, fundamental mechanisms underlying the delivery efficacy of ultrasound-targeted MB cavitation (UTMC) are poorly understood. Without this knowledge, the potential for UTMC to overcome many of the cellular barriers to bedside RNA therapeutics will not be realized. Accordingly, this proposal utilizes 2 distinct MB formulations and RNA payloads to systematically, for the first time, perform studies spanning individual cell signaling pathways, in vivo MB acoustic behaviors, and three-dimensional tissue interrogation of UTMC effects in vivo, to develop a cohesive paradigm addressing the mechanisms of UTMC-mediated endothelial hyperpermeability leading to RNA delivery. We hypothesize that MBs cavitating in the microcirculation mechanically perturb endothelial cells, leading to signaling events that culminate in endothelial barrier hyperpermeability and enhanced payload uptake. Using model systems, we propose in vitro studies to interrogate mechanistic pathways, then in vivo studies investigating UTMC endothelial barrier effects in real time, with 3 Aims: (1) Determine mechanisms by which UTMC increases endothelial barrier permeability. We will use endothelialized transwells and manipulate candidate pathways to test the hypothesis that UTMC-induced Ca2+ influx increases endothelial permeability, and optically measure attendant cellular events (multicolor confocal microscopy), correlating barrier function to cell response. (2) Determine the relationship between in vivo MB behaviors and transendothelial transport of siRNA using a custom ultrafast camera to visualize microvascular MB vibrations in vivo, testing the hypothesis that UTMC causes quantifiable mechanical events, then deriving physical principles governing UTMC-mediated hyperpermeability (3) Determine extravasation pathways and cellular fate of RNA-loaded MBs during UTMC in vivo using intravital high-speed multicolor confocal microscopy in cremaster microcirculation. Our multidisciplinary team unites physics/acoustics with biology/physiology to derive insights into biophysical mechanisms of UTMC-facilitated RNA delivery. Ultimately, our research will define a rational basis to optimize this remarkable technology, and hence accelerate the translation of RNA-based therapeutics to the bedside.